Project Summary/Abstract Idiopathic pulmonary fibrosis (IPF) impairs respiration through scarring of the interstitial space, resulting in (1) a stiffened lung that prevents inhalation and (2) limited gas exchange to adjacent pulmonary vasculature. Patients rely on lung transplants to extend their life beyond the median 3-year survival post-diagnosis as the only therapeutic agents used for IPF treatment slow disease progression are unable to cease or reverse fibrosis. The deficiency of pharmaceutical treatment strategies coupled with poor lung transplant survival rates indicates an obvious need for development of effective treatment options. Unfortunately, drug discovery has largely been restricted by physiologically irrelevant animal models and by reagent exhaustive, minimally informative in vitro platforms. The main goal of this proposal is to demonstrate our ability to identify pathways of importance in fibrotic progression and, by probing the IPF disease space, ultimately aid in therapeutic development. The outlined methodology enables biological discovery by systematically probing heterogenous populations of IPF cells by investigating functional behaviors of fibrotic cells from two complementary approaches. The first proposed methodology stratifies cell populations prior to encapsulation in collagen hydrogels to enable studies on how specific surface markers effect fibrotic behavior. Using flow sorting techniques, primary fibroblasts derived from IPF patients are separated into high and low expressing populations of specific surface receptors, a method that enhances the rarity and value of these precious cells. Stratified fibroblasts are then encapsulated in miniature collagen hydrogels using reagent-efficient microfluidic devices, reducing total cell volumes required for each condition and maximizing the number of surface receptors that can be investigated from a given cell source. Effects of low and high receptor expression will then be quantified and compared using several metrics of fibrotic function previously optimized. The second approach identifies potential therapeutic targets by functionally sorting and sequencing single fibrotic cells based on their contractility. Single fibroblasts are encapsulated into the previously described microgels and cultured to allow for spreading, adhering, and contracting of the cells into the surrounding matrix. As the cells exert force on the nearby collagen fibers, they compact the gel into a smaller sphere. Due to heterogeneity in the contraction ability of these fibroblasts, constructs are greatly varied in size and size-based sorting mechanisms are employed to segregate the most and least contractile cells. RNA sequencing is then used to directly compare transcriptomic profiles to fibroblast contractility and to identify upregulated pathways of interest. To validate sequencing hits as important mechanisms driving fibrotic functions, function-blocking antibodies and pharmaceutical inhibitors are used to confirm loss in fibrotic function for fibrogenic subpopulations.